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. 2014 Oct;128(4):525-41.
doi: 10.1007/s00401-014-1286-y. Epub 2014 May 8.

C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD

Elaine Y Liu  1 , Jenny Russ, Kathryn Wu, Donald Neal, Eunran Suh, Anna G McNally, David J Irwin, Vivianna M Van Deerlin, Edward B Lee
Affiliations

C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD

Elaine Y Liu et al. Acta Neuropathol. 2014 Oct.

Abstract

Hexanucleotide repeat expansions of C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal degeneration. The mutation is associated with reduced C9orf72 expression and the accumulation of potentially toxic RNA and protein aggregates. CpG methylation is known to protect the genome against unstable DNA elements and to stably silence inappropriate gene expression. Using bisulfite cloning and restriction enzyme-based methylation assays on DNA from human brain and peripheral blood, we observed CpG hypermethylation involving the C9orf72 promoter in cis to the repeat expansion mutation in approximately one-third of C9orf72 repeat expansion mutation carriers. Promoter hypermethylation of mutant C9orf72 was associated with transcriptional silencing of C9orf72 in patient-derived lymphoblast cell lines, resulting in reduced accumulation of intronic C9orf72 RNA and reduced numbers of RNA foci. Furthermore, demethylation of mutant C9orf72 with 5-aza-deoxycytidine resulted in increased vulnerability of mutant cells to oxidative and autophagic stress. Promoter hypermethylation of repeat expansion carriers was also associated with reduced accumulation of RNA foci and dipeptide repeat protein aggregates in human brains. These results indicate that C9orf72 promoter hypermethylation prevents downstream molecular aberrations associated with the hexanucleotide repeat expansion, suggesting that epigenetic silencing of the mutant C9orf72 allele may represent a protective counter-regulatory response to hexanucleotide repeat expansion.

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Figures

Fig. 1
Fig. 1
Hypermethylation of the C9orf72 promoter. a Cerebellar DNA from control (n = 8, left) and repeat-expanded cases (n = 8, right) were mock digested (no enzyme) or digested with MspI, HpaII or MspJI. DNA was subject to repeat primed PCR and representative electropherograms are shown. b Top panel shows a schematic of the bisulfite sequenced regions where filled boxes are exons, open boxes are CpG islands, and the star is the GGGGCC repeat expansion. Amplicon A covers the first CpG island, amplicon B covers the first half of the second CpG island and amplicon C covers the second half of the second CpG island. The bottom panels are summaries of bisulfite cloning results in which cerebellar DNA from four C9orf72 repeat expansion carriers and four control cases (n = 20–21 clones per genotype) was sequenced. Each oval represents a single CpG dinucleotide where unfilled oval represents an unmethylated CpG dinucleotide (0–10 % of clones) and a filled oval represents a methylated CpG dinucleotide (10–25 % of clones). Methylation over 25 % was not observed. c Top panel shows a schematic of the 5′ end of C9orf72 including the differentially methylated region (DMR, shaded) upstream of the 1st coding exon (E1) of C9orf72. The dinucleotide deletion polymorphism (rs200034037) and HhaI/HpaII cut sites are shown as arrows and the star is the hexanucleotide repeat expansion upstream of the 2nd coding exon (E2). DNA from C9orf72 promoter hypermethylated repeat-expanded cases (n = 3) that contain the polymorphism was mock digested (no enzyme) or digested with HpaII and HhaI. DNA from case 1 is from a lymphoblastoid cell line (ND14442) while DNA from cases 2 and 3 is from peripheral blood. The region flanking the deletion and restriction enzyme cut sites were amplified and run on a polyacrylamide gel to separate the major vs. minor rs200034037 alleles. Representative sequencing chromatograms of mock digested or HhaI/HpaII-digested DNA are shown where the gray area denotes the sequences demonstrating monoallelic vs. biallelic sequences downstream of rs200034037
Fig. 2
Fig. 2
C9orf72 promoter hypermethylation in repeat expanded and control brain. a Schematic representation of the 5′ end of the C9orf72 gene in which individual CpG dinucleotides are designated by vertical bars, the upstream CpG island is designated with an open box, the TSS for V2 and V3 transcripts are designated by arrows, and the hexanucleotide repeat region is designated by a star. The differentially methylated region (DMR) is shaded. The HhaI restriction enzyme recognition site in the differentially methylated region is shown. b Representative qPCR amplification curves for mock (no enzyme) versus HhaI-digested DNA demonstrating a shift in the amplification curve upon DNA digestion. The magnitude of this shift is used to calculate the % DNA resistant to HhaI digestion as a measure of DNA methylation. c DNA from control LCLs was in vitro methylated with MSssl, and various ratios of methylated and mock methylated DNA were tested. Digest qPCR quantification for HhaI resistance was plotted for increasing amounts of in vitro methylated DNA input. d HhaI digest resistance as assessed by digest qPCR of frontal cortex (n = 8–17) or cerebellum (n = 8–20) DNA from control (circles) or repeat expansion cases (squares) is shown. Individual values are plotted in addition to the mean and standard error. Two-way ANOVA: genotype p = 0.0009, region p = 0.8851, interaction p = 0.6445
Fig. 3
Fig. 3
C9orf72 methylation inhibits expression of mutant RNA. a ENCODE CAGE-seq quantification of C9orf72 mRNA. The total number of C9orf72 sequence tags was normalized for number of total sequence reads, shown as mean log2 transformed tags per million (TPM) ± SE. Cell lines were divided into different cell lineages as labeled, with other representing various mesenchymal and embryonic stem cell lineages. b ENCODE CAGE-seq quantification of C9orf72 V2 mRNA relative to total C9orf72 mRNA expression. The number of V2 tag sequences was normalized to total C9orf72 tag sequences, shown as mean % of total ± SE. c Southern blot of LCL DNA from non-expanded (ND16183) and expanded (ND11836, ND10966 and ND14442) cultures using a probe specific for C9orf72 that recognizes the normal allele (bottom) and expanded alleles (top). Molecular weight markers are shown as indicated. d HhaI resistance from non-expanded (ND16183) and expanded (ND11836, ND10966 and ND14442) LCLs, shown as mean + SE. One-way ANOVA: p < 0.0001. ****p < 0.0001 relative to ND16183. Each cell line was measured in triplicate. e–h RT-qPCR quantification shown as mean + SE (n = 3–5) for total mRNA (e), V2 mRNA (f), V3 mRNA (g) and intronic RNA (h) from control (left) or expanded (right) lymphoblast cells. *p < 0.05, **p < 0.01, ****p < 0.0001
Fig. 4
Fig. 4
C9orf72 promoter demethylation promotes toxic RNA accumulation. a HhaI resistance shown as mean + SE (n = 5) from control (left) or expanded (right) lymphoblast cells treated with 5-aza-dC (open) or untreated (filled). Two-way ANOVA: genotype p < 0.0001, treatment p < 0.0001, interaction p < 0.0001. ****p < 0.0001. b–e RT-qPCR quantification shown as mean + SE (n = 5) for total mRNA (b), V2 mRNA (c), V3 mRNA (d) and intronic RNA (e) from control (left) or expanded (right) lymphoblast cells treated with 5-aza-dC (open) or untreated (filled). Two-way ANOVA for total (genotype p = 0.0087, treatment p = 0.0264, interaction p = 0.0681), V2 (genotype p < 0.0001, treatment p = 0.5197, interaction p = 0.3579), V3 (genotype p = 0.026, treatment p = 0.0006, interaction p = 0.0076) and intronic RNA (genotype p = 0.0001, treatment p = 0.0259, interaction p = 0.0397). *p < 0.05, **p < 0.01. f–g Control (f) and expanded cells (g) that were untreated (filled) or 5-aza-dC treated (open) were assessed for toxicity (%LDH release relative to untreated, mean + SE, n = 4–8) after 24 h of no additional treatment (left), arsenite (10 μM, middle) or chloroquine (100 μM, right). Two-way ANOVA for control cells (5-aza-dC p = 0.0358, stressor p < 0.0001, interaction p = 0.0773) and mutant cells (5-aza-dC p < 0.0001, stressor p < 0.0001, interaction p = 0.1471). *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 5
Fig. 5
C9orf72 promoter hypermethylation inhibits RNA foci accumulation. a Representative in situ hybridization images of non-expanded (ND16183), unmethylated and expanded (ND11836), and hypermethylated and expanded (ND10966 and ND14442) LCLs. RNA foci in green are highlighted with arrows, and nuclei are counterstained blue with DAPI. Scale bar 10 μm. b Representative in situ hybridization images of non-expanded (left), hypomethylated and expanded (middle), and hypermethylated and expanded (right) cerebellar nuclei. RNA foci in green are highlighted with arrows, and nuclei are counterstained blue with DAPI. Arrowhead points to non-specific autofluorescence. Scale bar 5 μm. c The number of RNA foci in LCLs was scored (n = 200 cells per cell line), shown as a stacked bar graph to demonstrate the proportion of LCLs with zero, one or multiple RNA foci. d The percentage of cerebellar nuclei with RNA foci from 12 repeat-expanded cases is shown as a function of C9orf72 methylation. A linear regression was performed (R2 = 0.4633, p = 0.0148)
Fig. 6
Fig. 6
C9orf72 promoter hypermethylation and RANT accumulation in C9orf72 repeat expansion carriers. a Representative immunohisto-chemistry of cerebellar granular neurons using antibodies recognizing glycine–alanine (GA), glycine–proline (GP) and glycine–arginine (GR) dipeptide repeat proteins. Aggregates appear brown while nuclei are counterstained with hematoxylin. Scale bar 10 μm. b Quantification of RANT pathology in 11 hypomethylated and 7 hypermethylated C9orf72 repeat expansion carriers shown as mean + standard error of the percentage of cerebellar granular neurons containing GA (left), GP (middle) or GR (right) aggregates. Two-way ANOVA: p < 0.0001 (pathology type), p = 0.0080 (methylation), 0.0002 (interaction). c Sarkosyl-insoluble material was biochemically extracted and subject to dot blot analysis using antibodies that recognize GA, GP or GR dipeptide repeat proteins in five non-expanded controls (top row), five hypermethylated C9orf72 repeat expansion carriers (middle row), and five hypomethylated C9orf72 repeat expansion carriers (bottom row)
Fig. 7
Fig. 7
C9orf72 promoter methylation model. C9orf72 repeat expansions are associated with promoter hypermethylation in subset of mutation carriers. Expression of the repeat expansion leads to the accumulation of mutation-specific pathologies, namely RNA foci and DPR aggregates. The accumulation of mutant RNA is associated with increased vulnerability to cellular stressors, including oxidative and autophagic stress. Promoter hypermethylation inhibits these downstream effects by reducing the accumulation of RNA foci and/or DPR aggregates
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